7. Project Summary/Abstract Establishing and maintaining the necessarily distinctive protein complements of various intracellular membrane-bounded compartments depends upon dynamic tubulovesicular trafficking. Clathrin-coated vesicles are ubiquitous, short-lived, ~100-nm transport vesicles that play a fundamental role in defining the minute-by- minute composition of the eukaryotic plasma membrane. Identified over 50 years ago in electron micrographs by virtue of their characteristic polyhedrlal morphology, much information on their structure and function exists. A large catalogue of discrete components participate in the construction of the deceptively simple outer polyhedral clathrin lattice. Yet there remains much we do not currently comprehend about these vesicles, and arguably the most poorly understood aspect is their genesis. No consensus exists on what distinctly demarcates a membrane site where coat assembly will begin. Central issues to be resolved include precisely which proteins constitute the starter set of coat components, and what exactly these early-arriving so-called ?pioneers? contribute to the incipient assemblage. To associate productively with the plasma membrane, the principal inner layer component, the AP-2 adaptor complex, binds synchronously to phosphatidylinositol 4,5- bisphosphate and sorting signals displayed on the cytosolic portion of transmembrane cargo proteins. Yet, the cytosolic pool of AP-2 is in a closed conformation, disfavoring the simultaneous engagement of lipid, cargo and clathrin. We have shown that a functional three-protein nanocluster of AP-2 and the pioneers EPS15 and FCHO1/2 promotes conformational rearrangement of AP-2 to the open state, compatible with stable membrane deposition. The nucleating capacity of this nanocluser depends on the intrinsically unstructured C- terminal domain of EPS15, which binds to FCHO1/2, and on the disordered interdomain linkers in FCHO1/2, which bind to AP-2. An overarching theme of this proposal is to elucidate how unstructured stretches of amino acids in endocytic pioneers contribute to clathrin-coat nucleation. Based on biochemical and atomic structural information, a model for the focal structural reorganization of AP-2 within the plasma-membrane docked nanocluster will be scrutinized. The role of casein kinase 2-mediated phosphorylation of the FCHO2 linker in binding to AP-2 will be evaluated and followed up with cell-based and in vitro liposome reconstitution studies. Novel single chain llama nanobodies against EPS15, FCHO2 and CALM will be classified and applied to the study the role of allovalency and fuzzy binding phenomena during nanocluster formation and coat nucleation. Finally, the contribution of localized liquid-liquid phase separation, driven by intrinsically disordered regions in the early-arriving cohort of coat proteins will be examined. The key hypothesis is that assemblies of scaffolding proteins in a demixed, gel-like condensate both concentrate and exclude reactants at the nascent bud to promote directional biochemical reactions. The concepts are innovative with broad potential impact to understanding and further studying intracellular protein trafficking.